Isolation, purification and identification of ellagic acid derivatives, catechins, and procyanidins from the root bark of Anisophyllea dichostyla R. Br.

The root bark of Anisophyllea dichostyla R. Br. is traditionally used in the Democratic Republic Congo for the treatment of several conditions such as anorexia, fatigue and intestinal infections. We have identified and quantitated several polyphenol antioxidants in the methanol extract of the root bark (120g). The polyphenol content (3.32g/kg) was predominantly ellagitannins (25%) and polyhydroxyflavan-3-ols (catechins and procyanidins, 75%) with 3'-O-methyl-3,4-methylenedioxo ellagic acid 4'-O-beta-d-glucopyranoside and (-)-epicatechin as the major species in each class. These two compounds and the following species were identified unequivocally by NMR spectroscopy: (+)-catechin, (-)-epicatechin 3-O-gallate, 3-O-methyl ellagic acid, 3,3'-di-O-methyl ellagic acid, 3'-O-methyl-3,4-methylenedioxo ellagic acid, 3'-O-methyl-3,4-methylenedioxo ellagic acid 4'-O-beta-d-glucopyranoside, and 3'-O-methyl ellagic acid 4-O-beta-d-xylopyranoside. The following additional compounds were purified by semi-preparative HPLC and tentatively identified on the basis of UV spectra, HPLC-ESI-MS and nano-ESI-MS-MS: (+)-catechin-3-O-beta-d-glucopyranoside, epicatechin-(4beta-->8)-catechin (procyanidin B(1)), epicatechin-(4beta-->8)-epicatechin (procyanidin B(2)), an (epi)catechin trimer, 3-O-methyl ellagic acid 4-O-beta-d-glucopyranoside, (-)-epicatechin 3-O-vanillate, 3,4-methylenedioxo ellagic acid 4'-O- beta-d-glucopyranoside, and 3,3'-di-O-methyl ellagic acid 4-O-beta-d-xylopyranoside. Fractionation of the raw extract by column chromatography on silicic acid yielded 10 fractions. In the hypoxanthine/xanthine oxidase antioxidant assay system, CC-9 which contained a range of polyphenols dominated by (-)-epicatechin-O-gallate proved to be the most potent antioxidant fraction (IC(50)=52 micro g/mL) in terms of ROS scavenging. In terms of XO inhibition CC-8, dominated by (epi)catechin trimer and which also contained appreciable amounts of 3'-O-methyl ellagic acid 4'-O-beta-d-xylopyranoside, as well as the catechins (+)-catechin-3-O-beta-d-glucopyranoside, epicatechin-(4beta-->8)-catechin (procyanidin B(1)), and (-)-epicatechin 3-O-gallate, proved to be the most potent (IC(50)=36 micro g/mL).     

Antioxidant activity of tannin components from Vaccinium vitis-idaea L.

Reactive oxygen molecules have been implicated as important pathological mediators in many clinical disorders and periodontal disease. To provide possible alternative treatment of periodontal disease, six tannins isolated from Vaccinium vitis-idaea L. were evaluated for anti-lipid peroxidation, anti-superoxide formation and free radical scavenging activity. The results showed that cinnamtannin B1 displayed the strongest anti-lipid peroxidation activity, proanthocyanidin A-1 displayed the strongest superoxide scavenging activity, and epicatechin-(4beta--> 6)-epicatechin-(4beta-->8, 2beta-->O--> 7)-catechin had the strongest anti-superoxide formation effect. We conclude that tannins isolated from V. vitis-idaea L. exhibited multiple antioxidant activity, and could be used for the treatment of periodontal disease.     

Antimicrobial activity of tannin components from Vaccinium vitis-idaea L

Reactive oxygen species have been implicated as important pathological mediators in many clinical disorders, including periodontal disease. As a possible alternative for the treatment of periodontal disease, the antimicrobial activity of six tannins isolated from Vaccinium vitis-idaea L., with confirmed antioxidant activity, were assayed by the agar dilution method against selected periodontal pathogens, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia. The results showed that epicatechin-(4beta-->8)-epicatechin-(4beta-->8, 2beta-->O-->7)-catechin had strong antimicrobial activity against P. gingivalis and P. intermedia, but not A. actinomycetemcomitans. The other tannins tested did not show antimicrobial activity. We conclude that tannins isolated from V. vitis-idaea L. with antimicrobial activity could potentially be used for the treatment of periodontal disease.     

Procyanidins from the herb of Hypericum perforatum.

From the aqueous acetone extract of the herb of Hypericum perforatum the flavanols catechin (1) and epicatechin (2), and the procyanidins A2 (9), B1 (3), B2 (4), B3 (5), B5 (6), B7 (7) and C1 (8) were isolated. Their structures were established as their peracetate derivatives, on the basis of chemical and spectral evidence. The 13C NMR spectrum of the higher molecular weight polymer fraction revealed a 3',4'-dihydroxylated B-ring oxidation pattern and the 2,3-cis relative stereochemistry of the constituent flavan-3-ol units. The mean average molecular size of the polymers was estimated to be 4 to 5 flavan-3-ol units. The procyanidin pattern in comparison to that of Crataegus spec. is briefly discussed.     

Phenolic compounds on the leaves ofBetula platyphylla var.latifolia

Chemical examination ofBetula platyphylla var. latifolia afforded a novel diarylheptanoid named betulatetraol, together with a phenylpropanoid (3,4′-dihydroxypropiophenone), flavan-3-ol [(+)-catechin] and its glycosides [(+)-catechin 5-O-β-D-glucopyranoside, (+)-catechin 7-O-β-D-glucopyranoside] and two proanthocyanidins (procyanidins B-1 and B-3).     

Proliferative effects of flavan-3-ols and propelargonidins from rhizomes of<Emphasis Type="Italic">Drynaria fortunei</Emphasis> on MCF-7 and osteoblastic cells

The proliferative effects of thirty Oriental medicinal herbs on MCF-7 (estrogen-sensitive breast cancer cell line) and ROS 17/2.8 osteoblast-like cells were determined using the MTT assay. Methanol extracts from several herbs was found to show proliferative activity on the above two cell lines in the range of 5 to 100 microg/mL. Among these active herbs, the methanol extract from the rhizomes of Drynaria fortunei showed the most potent proliferative activity, and the cell proliferations were significantly increase by 136 and 158% in the MCF-7 and ROS 17/2.8 cells, respectively, when treated with 100 microg/mL. Through a bioassay-guided separation, eight flavonoids, including four new flavan-3-ols and two propelargonidins, together with the known (-)-epiafzelechin and naringin, were isolated. Their chemical structures were characterized as (-)-epiafzelechin (1), (-)-epiafzelechin-3-O-beta-D-allopyranoside (2), (-)-epiafzelechin-3-O-(6"-O-acetyl)-beta-D-allopyranoside (3), 4beta-carboxymethyl-(-)-epiafzelechin methyl ester (4), 4beta-carboxymethyl-(-)-epiafzelechin sodium salt (5), naringin (6), (-)-epiafzelechin-(4beta-->8)-4beta-carboxymethylepiafzelechin methyl ester (7) and (-)epiafzelechin-(4beta-->8, 2beta-->O-->7)-epiafzelechin-(4beta-->8)-epiafzelechin (8) by extensive 1D and 2D NMR spectroscopy. Most of these flavonoids, in the range of 10(-15) to approximately 10(-6) M, accelerated the proliferation of MCF-7 cell, with compounds 7 and 8, in the range of 10(-15) to approximately 10(-12) M, showing especially potent proliferation effects. Meanwhile, seven flavonoids, with the exception of compound 4, stimulated the proliferation of ROS 17/2.8 cells in the range of 10(-15) to approximately 10(-6) M, with compounds 5-8 especially accelerating the proliferation, in dose-dependent manners (10(-15) to approximately 10(-9) M), and their proliferative effect was much stronger than that of E2 and genistein. These results suggest that propelargonidin dimers and trimers isolated from the rhizomes of Drynaria fortunei may be useful as potential phytoestrogens, which play important physiological roles in the prevention of postmenopausal osteoporosis.     

Quantification of procyanidins in oral herbal medicinal products containing extracts of Crataegus species

According to the European Pharmacopeia a photometric assay is used for the estimation of procyanidins in Crataegi fructus. This assay is also most commonly used for procyanidin analysis in herbal medicinal products (HMPs) containing extracts of hawthorn (Crataegus species). In order to find an appropriate method for the determination of oligomeric and polymeric procyanidins by analysing various preparations containing extracts of Crataegus, the Ph. Eur.-method was compared to an HPLC-method with chemical reaction detection (HPLC-CRD-method) and another conventional photometric assay using 4-dimethylamino-cinnamic-aldehyde (DMACA). Total procyanidins estimates obtained with the pharmacopeial method were, depending on the reference standard used, at least more than 50% higher than those obtained with the DMACA-assay. The determination of individual procyanidins could only be achieved by HPLC-CRD. Monomeric, dimeric, and trimeric procyanidins could be separated and detected individually, whereas no HPLC separation was possible for higher polymeric compounds. However, these compounds could be analysed as co-eluting groups. Using the DMACA method for the estimation of total oligomeric procyanidins and the HPLC-CRD method for quantification of the mono- up to trimeric procyanidins, some market leading herbal medicinal products from Germany containing extracts Crataegus species (C. monogyna Jacq., C. laevigata D.C., C. pentagyna Waldst. et Kit., C. nigra Waldst. et Kit, C. azarolus L.) were analysed. Procyanidin B2 (epicatechin-(4 beta-->8)-epicatechin) was isolated from Aesculus hippocastanum fruit shells as reference standard for calibration purposes. The structure elucidation was carried out by by means of MS and 1H-NMR. Quantitative 1H-NMR spectroscopy (qNMR) was applied for purity assessment.     

Oligomeric proanthocyanidins from Rumex acetosa L. inhibit the attachment of herpes simplex virus type-1

The polyphenole-enriched acetone-water extract R2 from the aerial parts of Rumex acetosa L. containing high amounts of oligomeric and polymeric proanthocyanidins and flavonoids was tested for antiviral activity. R2 exhibited strong antiviral activity against herpes simplex virus type-1 (HSV-1) while the replication of adenovirus 3 was not affected. By plaque reduction test and MTT assay on Vero cells, the HSV-1-specific inhibitory concentration (IC(50)) and cytotoxic concentration (CC(50)) were determined. R2 exibited an IC(50) of 0.8 μg/mL and a selectivity index (SI) (ratio of IC(50) to CC(50)) of approximately 100 when added to the virus inoculum for 1h at 37°C prior to infection. The antiviral activity was due to the presence of flavan-3-ols and oligomeric proanthocyanidins in the extract. Structure-activity analyses indicated that flavan-3-ols and proanthocyanidins with galloylation at position O-3 are highly potent compounds (SI>40), while ungalloylated compounds did not exhibit antiviral effects (SI<1). R2 and a major proanthocyanidin from R2, epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate abolished virus entry into the host cell by blocking attachment to the cell surface. When added after attachment at a concentration of ≥ 12.5 μg/mL, R2 inhibited also penetration of HSV-1 into the host cell. R2 and epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate were shown to directly interact with viral particles leading to the oligomerisation of envelope proteins as demonstrated for the essential viral glycoprotein gD. Using raft cultures with three-dimensional organotypic human skin equivalents it was shown that treatment of cultures with R2 after infection with HSV-1 resulted in a reduced viral spread.     

An analysis method for flavan-3-ols using high performance liquid chromatography coupled with a fluorescence detector

Procyanidins belong to a family of flavan-3-ols, which consist of monomers, (+)-catechin and (−)-epicatechin, and their oligomers and polymers, and are distributed in many plant-derived foods. Procyanidins are reported to have many beneficial physiological activities, such as antihypertensive and anticancer effects. However, the bioavailability of procyanidins is not well understood owing to a lack of convenient and high-sensitive analysis methods. The aim of this study was to develop an improved method for determining procyanidin content in both food materials and biological samples. High performance liquid chromatography (HPLC) coupled with a fluorescence detector was used in this study. The limits of detection (LODs) of (+)-catechin, (−)-epicatechin, procyanidin B2, procyanidin C1, and cinnamtannin A2 were 3.0 × 10⁻³ ng, 4.0 × 10⁻³ ng, 14.0 × 10⁻³ ng, 18.5 × 10⁻³ ng, and 23.0 × 10⁻³ ng, respectively; the limits of quantification (LOQs) were 10.0 × 10⁻³ ng, 29.0 × 10⁻³ ng, 28.5 × 10⁻³ ng, 54.1 × 10⁻³ ng, and 115.0 × 10⁻³ ng, respectively. The LOD and LOQ values indicated that the sensitivity of the fluorescence detector method was around 1000 times higher than that of conventional HPLC coupled with a UV-detector. We applied the developed method to measure procyanidins in black soybean seed coat extract (BE) prepared from soybeans grown under three different fertilization conditions, namely, conventional farming, basal manure application, and intertillage. The amount of flavan-3-ols in these BEs decreased in the order intertillage > basal manure application > conventional farming. Commercially available BE was orally administered to mice at a dose of 250 mg/kg body weight, and we measured the blood flavan-3-ol content. Data from plasma analysis indicated that up to the tetramer oligomerization, procyanidins were detectable and flavan-3-ols mainly existed in conjugated forms in the plasma. In conclusion, we developed a highly sensitive and convenient analytical method for the analysis of flavan-3-ols, and applied this technique to investigate the bioavailability of flavan-3-ols in biological samples and to measure flavan-3-ol content in food material and plants.     

Flavan-3-ols from Ulmus davidiana var. japonica with inhibitory activity on protein glycation

Four flavan-3-ols, (+)-catechin ( 1), (+)-catechin 7- O- beta- D-apiofuranoside ( 2), (+)-catechin 7- O- beta- D-xylopyranoside ( 3), (+)-catechin 7- O- beta- D-glucopyranoside ( 4), and proanthocyanidin A-1 ( 5) as well as three other constituents, isolated from an EtOAc-soluble extract of the stem barks of Ulmus davidiana var. japonica, were evaluated for inhibitory activity against the formation of AGEs. Compounds 1 - 5 exhibited a significant inhibitory activity on the formation of AGEs in an AGEs-BSA assay by specific fluorescence and this was confirmed by an indirect AGEs-ELISA. Moreover, compounds 1 and 5 markedly reduced AGEs-BSA cross-linking to collagen in a dose-dependent manner. AGEs:advanced glycation end products BSA:bovine serum albumin CC:column chromatography CD:circular dichroism.     

Isolation of two new bioactive proanthocyanidins from Cistus salvifolius herb extract

Two new proanthocyanidins, epigallocatechin-3-O-p-hydroxybenzoate-(4beta-->8)-epigallocatechin (1) and epigallocatechin-3-O-p-hydroxybenzoate-(4beta-->8)-epigallocatechin-3-O-gallate (2) in addition to the known compound epigallocatechin-(4beta-->6)-epigallocatechin-3-O-gallate (3), were isolated from the air-dried herb of Cistus salvifolius. The chemical structures were determined on the basis of 1D-and 2D-NMR-spectra (HSQC, HMBC) of their peracetylated derivatives, MALDI-TOF-mass spectra, and by acid-catalysed degradation with phloroglucinol. The isolated compounds 1-3 and the water extract of C. salvifolius herb were tested for their inhibitory activities against COX-1 and COX-2. Compound 2 showed the strongest inhibitory effect on COX-2 followed by compound 3, compound 1 and the water extract, while compounds 1-3 exhibited moderate in vitro inhibition against COX-1.     

Methylation of catechins and procyanidins by rat and human catechol-O-methyltransferase: metabolite profiling and molecular modeling studies

Catechins and procyanidins are major polyphenols in plant-derived foods. Despite intensive studies in recent years, neither their biochemical nor their toxicological properties have been clarified sufficiently. This study aimed to compare the methylation of catechins and procyanidins by the enzyme catechol-O-methyltransferase (COMT) in vitro. We conducted incubations with rat liver cytosol and human placental cytosol including S-adenosyl-l-methionine. The set of substrates comprised the catechins (-)-epicatechin (EC) and (+)-catechin (CAT), the procyanidin dimers B1, B2, B3, B4, B5, and B7 as well as procyanidin trimer C1. After extraction, metabolites were analyzed by means of liquid chromatography-electrospray ionization-mass spectrometry and liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. EC and CAT were converted to two monomethylated metabolites each by human and rat COMT, with the 3'-O-methyl derivatives being consistently the main metabolites. Furthermore, the flavanyl units of procyanidins were methylated consecutively, leading to monomethylated and dimethylated dimeric metabolites as well as monomethylated, dimethylated, and trimethylated C1 metabolites. The methylation status of each flavanyl unit was determined by means of mass spectrometric quinone-methide fragmentation patterns. In addition, molecular modeling studies were performed with the aim to predict the preferred site of methylation and to verify the experimental data. In conclusion, our results indicate that the degree and position of methylation depend clearly on the three-dimensional structure of the entire substrate molecule.     

Geranins C and D, additional new antiprotozoal A-type proanthocyanidins from Geranium niveum

Two new additional A-type proanthocyanidins have been isolated from Geranium niveum. Their structures were determined by spectroscopic, chemical and chiroptical methods as epi-afzelechin-(4beta-->8,2beta-->O-->7)-gallocatechin (1) and epi-afzelechin-(4beta-->8,2beta-->O-->7)-afzelechin-(4beta-->8,2beta-->O-->7)-afzelechin (2). Proanthocyanidins 1 and 2 were given the trivial names of geranins C and D, respectively. Compound 2 showed moderate antiprotozoal activity against Entamoeba histolytica and Giardia lamblia, whereas 1 exhibited weak activity toward E. histolytica.     

Towards the synthesis of proanthocyanidins: half a century of innovation

Results emanating from the synthesis of proanthocyanidins played a crucial role in defining the constitution, regiochemistry, and absolute configuration of this complex but fascinating group of plant secondary metabolites. The initial efforts, commencing in 1966, were focused on structure elucidation of, especially, the procyanidins, profisetinidins, and prorobinetinidins. However, over the past 12 years the emphasis has shifted to the synthesis of the bioactive procyanidins and some of their derivatives at a scale that would permit assessment of their pharmacological properties. With a few exceptions, the vast majority of these synthetic protocols involve the formation of the interflavanyl bond by acid/Lewis acid activation at C-4 of a flavan-3,4-diol or its equivalent, and subsequent trapping of the incipient C-4 carbocation by the nucleophilic centers of a flavan-3-ol (catechin). This review represents the first comprehensive chronicle depicting the development of the subject of proanthocyanidin synthesis.     

Antiplasmodial phenolic compounds from Piptadenia pervillei

Piptadenia pervillei Vatke (Fabaceae) was selected from a screening programme devoted to the search of naturally-occuring antimalarial compounds from plants of Madagascar. Bioassay-guided fractionation of the ethyl acetate extract of the leaves led to the isolation of four phenolic compounds, (+)-catechin ( 1), (+)-catechin 5-gallate ( 2), (+)-catechin 3-gallate ( 3) and ethyl gallate ( 4). Structures were determined by NMR and mass spectroscopy. Compounds 2 and 3 displayed the highest in vitro activity against the chloroquine-resistant strain FcB1 of Plasmodium falciparum with IC (50) values of 1.2 microM and 1.0 microM, respectively, and no significant cytotoxicity against the human embryonic lung cells MRC-5 was measured (IC (50) values > 75 microM). Five analogues ( 5 - 9) of (+)-catechin 5-gallate ( 2) were synthesized and evaluated for their antiplasmodial activity.     

A new flavan-3-ol from Artocarpus nitidus subsp. lingnanensis

Further investigation on the stems of Artocarpus nitidus subsp. lingnanensis led to the isolation and characterization of a new flavan-3-ol, named artoflavanocoumarin (1), along with three known compounds (+)-catechin (2), (+)-afzelechin 3-O-α-l-rhamnoside (3), and (+)-catechin 3-O-α-l-rhamnoside (4). Their structures were elucidated on the basis of spectroscopic data.     

Three new polyphenols from the rhizomes of Arachniodes exilis

Three new polyphenols, araspidin BB (1), arachniodesin A (2) and arachniodesin B (3), together with two known compounds, epicatechin (4) and procyanidin B-2 (5), were isolated from the rhizomes of Arachniodes exilis. The structures of three new compounds were elucidated as 5-methyl-methylene-bis-phlorobutyrophenone (1), 4beta-ethoxycarbonylmethylepicatechin (2) and epicatechin-(4beta --> 8)-4beta-ethoxycarbonylmethylepicatechin (3), on the basis of their spectral analysis and by comparing them with the related model compounds. Compounds 4 and 5 were obtained from the title plant for the first time.     

Inhibitory effects of Luobuma tea and its components against glucose-mediated protein damage.

Luobuma tea, prepared from the leaves of Apocynum venetum L., is a popular beverage in China. In this study, the activity of Luobuma leaf extract and its components against the formation of advanced glycation endproducts (AGEs), which are largely involved in the pathogenesis of diabetic vascular complications, was examined using the in vitro glycation reaction. Strong inhibitory activity against the formation of AGEs was shown by Luobuma aqueous extract. Following further fractionation of this extract, seven polyphenolic compounds, i.e. (+/-)-gallocatechin, (-)-epigallocatechin, (+/-)-catechin, (-)-epicatechin, epicatechin-(4beta-8)-gallocatechin, epigallocatechin-(4beta-8)-epicatechin and procyanidin B-2, were isolated by Sephadex LH-20 column chromatography. These purified compounds also exerted inhibitory activities that were more potent than the positive control, aminoguanidine. Our findings may help to explain the beneficial effects of this plant against atherosclerosis.     

Antioxidant protection of low density lipoprotein by procyanidins: structure/activity relationships

The antioxidant activity of catechins and oligomeric procyanidins against low density lipoproteins peroxidation was studied by means of three distinct methods: cis-parinaric acid fluorescence decay, conjugated-dienes detection, and oxygen consumption. A relationship between the radical trapping efficiency of procyanidins and their structure was investigated. The results indicated that: (i) interflavan linkage type (C4[bond]C6 or C4[bond]C8) exerts a significant effect upon radical-trapping antioxidant activity of procyanidins. It is suggested that the conformation adopted by each procyanidin in aqueous solution influence their hydrophilic character, hence affecting their interaction with the peroxyl radicals present in aqueous phase and those in LDL particle (lipidic nature); (ii) antioxidant activity increase with the degree of polymerization for the compounds with (-)-epicatechin (epi) as structural unit (epi, dimer B2 (epi-epi) and trimer C1 (epi-epi-epi)); (iii) galloylation increases antioxidant activity of procyanidins, specially in the case of B2-3"-O-gallate dimer, which revealed the maximal trapping efficiency.     

药用大黄中蒽醌和非蒽醌类成分的分离与鉴定

目的对药用大黄中蒽醌及非蒽醌类成分进行深入研究。方法药用大黄80%(φ)乙醇溶液提取物经硅胶柱、ODS柱、MCI gel CHP20、SephadexLH-20凝胶柱色谱及HPLC色谱分离,纯化,根据理化性质和波谱数据进行结构鉴定。结果分离得到6个蒽醌类和5个非蒽醌类成分,经波谱数据解析,分别鉴定为香兰基丙酮(vanillylacetone,1)、大黄素(emodin,2)、芦荟大黄素(aloeemodin,3)、大黄素甲醚-8-O-β-D-葡萄糖苷(physcion-8-O-β-D-glucopyranoside,4)、大黄酚-8-O-β-D-葡萄糖苷(chrysophanol-8-O-β-D-glucopyranoside,5)、1-甲基-8-羟基-9,10-蒽醌-3-O-β-D-(6'-O-桂皮酰基)吡喃葡萄糖苷(1-methyl-8-hydroxyl-9,10-anthraquinone-3-O-β-D-(6'-O-cinnamoyl)glucopyrano-side,6)、芦荟大黄素-3-(羟甲基)-O-β-D-葡萄糖苷(aloe emodin-3-(hydroxymethyl)-O-β-D-glucopy-ranoside,7)、(+)儿茶素(catechin,8)、表儿茶素没食子酸酯(epicatechin-3-O-gallate,9)、儿茶素-3-O-β-D-葡萄糖苷(catechin-3-O-β-D-glucopyranoside,10)、儿茶素-8-β-D-葡萄糖苷(catechin-8-β-D-glucopyranoside,11)。结论化合物1,6,9-11为首次从该植物中分离。